Submitted on November 8, 2006
Revised on April 18, 2007
Accepted on April 23, 2007
The transfer of very low density lipoprotein- associated phospholipids to activated human platelets depends upon cytosolic phospholipase A2 activity
Salam Ibrahim, Catherine Calzada, Valérie Pruneta-Deloche, Michel Lagarde, and Gabriel Ponsin
UMR 870 INSERM/INSA-Lyon, RMND, Régulations Métaboliques, Nutrition et Diabètes, Villeurbanne 69621
Corresponding Author: gabriel.ponsin{at}insa-lyon.fr
We have previously reported that VLDL could transfer phospholipids (PL) to platelets and that these transfers were favored by thrombin or lipoprotein lipase (LPL)- mediated platelet activation. The present work was undertaken to identify the platelet metabolic pathway involved in this process. The transfer of radiolabelled PL from VLDL (200 µM PL) to platelets (2x108/mL) was measured after incubations of 1 h at 37°C, with or without thrombin (0.1 U/mL) or LPL (500 ng/mL). To discriminate between metabolic pathways, various inhibitors, including aspirin, a cyclooxygenase inhibitor (300 µM), esculetin, a 12-lipoxygenase inhibitor (20 µM), Methyl-Arachidonyl-Fluorophosphonate (MAFP), a phospholipase A2 (PLA2) inhibitor (100 µM), 1,2-Bis (2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid tetrakis (acetoxymethyl) ester (BAPTA-AM), an intracellular Ca2+ chelator (20 µM), bromoenol lactone (BEL), a Ca2+ independent-PLA2 (iPLA2) inhibitor (100 nM), or 1-[6-[[17ß-3-Methoxyestra-1,3,5(10)-trien-17-yl-]amino]hexyl]-1H-pyrrole-2,5-dione (U73122), a phospholipase C (PLC) inhibitor (20 µM), were added to the incubation medium. Aspirin and esculetin had no effect, showing that PL transfer was not directly dependent upon cyclooxygenase or lipoxygenase pathways. The transfer of PL was inhibited by MAFP, U73122 or BAPTA-AM. Although MAFP inhibits both cytosolic PLA2 (cPLA2) and iPLA2, only cPLA2 is a calcium- dependent enzyme. Since intracellular calcium mobilization is favored by PLC and inhibited by BAPTA-AM, our data suggest that the transfer of PL from VLDL to platelets results from a cPLA2 rather than a iPLA2- dependent process. This conclusion was confirmed by our observation that the inhibition of iPLA2 by BEL had no effect on PL transfers.
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